A site where a man with far too much interest in beer gets to write about it.

Wednesday, 9 February 2011

More fun with a microscope

I've been a scientist again recently. My friend young Rob had phoned me asking for some tips on using a microscope so I went through a few of the things you can do with a microscope in a brewery and took some pictures.

Mostly microscopes will be used in a brewery as part of the yeast management, though they can have their place when investigating infection. The most basic thing you can do is have a look see at what's going on at the microscopic level. What could be easier than that? The thing to remember is that you'll be looking at something that's been magnified up to a 1000 times, so when taking your sample remember less is more.

First I took a microscope slide. It's worthwhile getting ones with frosted edges as these have a section that you can write on in pencil which makes labeling easier. I placed a small drop of water in the middle of the slide.

Here I've used a disposable plastic pasteur pipette or pastette. This drop had to have evaporated before I could look at the slide so I kept the drop small.

Next I took a sample of some yeast we'd harvested.

It's a bit out of focus but you can see on the end of the disposable plastic loop a small amount of yeast. This was more than enough.

I mixed the yeast in with the drop of water and spread it across the surface of the slide.

The water had a very slight milkiness when the yeast was mixed in.

Now the slide needed to be dried before it could be stained. Microorganisms are hard to see, even under a microscope, so stains are usually added to make them easier to see, and to some extent help identify them. The slide can be left on the bench to air dry or heat can be applied until the water has evaporated. Bunsen burners, with of course sufficient air to give a blue flame, are what will be used in a well equipped laboratory and Camping gaz bunsen burners are even available for where there are no gas taps. Small blow torches can also be used, though they are less stable. Heating over a candle or lighter will lead to soot collecting on the bottom of the slide which will need to be cleaned off with blue roll before the slide can be looked at. When heating a slide remember it's made of glass so don't hold it continuously over the flame, pass it slowly over the flame a number of times until it has dried.

Heating the slide has the benefit of fixing the sample to the slide. This means the sample is firmly attached to the glass, denatured, and ready to receive the stain. For this reason it's usual to heat a slide for a short while even if it's air dried. You should be able to skip this with brewery yeast if you apply the stain delicately though.

Having dried the slide I took it to the sink and flooded it with methylene blue stain. The stain was left on for a few seconds and then rinsed off with tap water.

The slide was left to dry again and this is what I ended up with:

Now we get to the microscope!

When a microscope is out of focus I start by using the x 10 objective lens. I racked the microscope stage right up and lowered the slide using the coarse focus until I could see the yeast. Well, at this stage all I could see were some patches of something blue so I moved round to the x 40 objective lens. I usually start with the light on full power and the diaphragm shut and start to open the diaphragm as I move up the magnifications.

At this point I used the fine focus to bring the yeast back into view. Once I could see the yeast again I moved the lens away and applied some immersion oil to the slide. This has the same refractive index as glass and cuts down on distortion at the higher magnifications. Then I swung round the x 100 lens so it was touching the oil.

At this magnification it can be a bit tricky to bring your sample into focus so I'll often close down the diaphragm a bit and start moving the slide slowly back and forth so I know to focus on a moving item and not try and focus on say, a speck of dust on the eye piece. Again it was only adjustments to the fine focus that were needed, try focusing one way and if that doesn't work going back the other way until the sample can be clearly seen.

This picture I ripped off the internet shows the sort of thing I was seeing at this point:

If bacteria were present they'd most likely be small rods, much smaller that the yeast.

Most often brewery based microscope fun involves doing yeast counts, which are a little more long winded. To do this I took 1 ml of yeast slurry.

And mixed it with 9 ml of water in a universal container giving me a 1 in 10 dilution.

But that's not dilute enough so I took 1 ml of the first dilution and added it to another 9 ml of water in another universal giving me a 1 in 100 dilution.

From the 1 in 100 dilution I then took 1 ml and mixed it with 1 ml of methylene blue stain.

Having got the yeast diluted and stained it was time to bring out the haemocytomer. This is a slide with a grid of known area etched into it and when the cover glass is attached this gives a known volume in which cells can be counted. As the name suggests they were originally used for counting blood cells, though that's mostly done with machines now, except in the case of birds and reptiles. As these animals have nucleated red blood cells some manual methods are still required and in a previous job I occasionally used a heamocytomer for this very purpose. But I digress.

A quick dab of spit either side of the counting grid helped stick the cover glass in place and then by carefully flooding the chamber our yeast was ready to count.

It was back to focusing the microscope again, though due to the thickness of the slide I could only go as far as the x 40 objective lens.

Using another picture ripped off the internet I can show you that I saw something like this:

The clear round objects are live yeast cells, the blue round objects are dead yeast cells and the amorphous blue blobs are debris which can be ignored. By counting the number of live cells and dead cells the % viability of the yeast can be established and a quick calculation can give you a cell count. After I'd left Heriot-Watt I emailed the bloke that works in the brewery there for his top tips and here are his words of wisdom:

"Hi Ed

Did you not learn anything in the last 9 months!!!!!

OK this is what I do.

Make a 1:100 W/W dilution of the yeast slurry with cold tap water.

Mix well, then mix 1 ml diluted yeast with 1ml methylene blue.

Put onto slide and count 5 sets of 16 small squares.

To calculate the yeast concentration:-

Divide your white cell count by 80 (the number of small squares counted) to give the average number of cells per small square.

Multiply the answer by 4 million then by 200 (the dilution factor).

This will then give you the concentration of cells per gram in the yeast slurry.

Hope this helps."

So that's what I did. And there I had it, a yeast count to go with my yeast viability. You can also see bacteria if they're present, though they won't be stained so are harder to spot. Not that there were any present in my yeast of course.